The long term objectives of this application are intended to establish the biochemical properties and functional significance of new lysosomal-type protease (endopeptidases and exopeptidases) recently detected in guinea pig epididymal spermatozoa. One of the endopeptidases resembles lysosomal cathepsin L, but is totally masked in epididymal sperm. A second cysteine proteinase (which may be responsible for unmasking "cathepsin L") has also been detected. This endopeptidase shares some properties with cathepsin S -- a poorly studied lysosomal cysteine proteinase thus far found only in lymphatic tissues. These two cathepsins are uniquely and selectively concentrated (to the exclusion of detectable levels of cathepsins B and H) in guinea pig sperm. The principal exopeptidase to be investigated resembles dipeptidyl peptidase II (DPP II), a lysosomal enzyme belonging to the serine catalytic class that is present at extraordinary levels in epididymal sperm. Efforts will be made to purify these proteases from freshly collected (guinea pig) epididymal sperm homogenates for the purpose of establishing their molecular properties, substrate specificity, reaction kinetics, inhibitor sensitivities, and immunological cross reactivity with their lysosomal counterparts. Purification methods will be employed that exploit differential solubilities, molecular size, charge, isoelectric points, hydrophobicity, and special affinities. In view of the limited supply of tissue available for this purification work, high performance adaptations of traditional methodologies will be utilized. These (FPLC) procedures, which are currently being employed in this laboratory, include ion (anion and cation) exchange chromatography, molecular exclusion chromatography, chromatofocusing, and hydrophobic interaction chromatography. Specific fluorometric assays together with azo-dye and fluorescence enzyme histochemical procedures will be employed (or developed) as needed to assay and to localize these "new proteases" during spermatogenesis and sexual development. The possible functional significant of latent cathespin L will be assessed in relation to capacitation, the acrosome reaction, and proacrosin activation. Insights gained from the proposed studies will contribute to a basic understanding of the role of acrosomal proteases in reproduction and facilitate a more rational approach to problems of contraception, unexplained, infertility, and in vitro fertilization.